Decades of research on the myriad of biological activities that the polyamines, putrescine, spermidine and spermine play in cellular processes have shown the profound role they play in life (Cohen, S. S., “A Guide to the Polyamines” 1998, Oxford University Press, New York). As polycations at physiological pH, they bind tightly to and strongly modulate the biological activities of all of the anioinic cellular components.
Many stimuli involved in both normal and neoplastic growth activate the polyamine biosynthetic pathway. A great number of multidisciplinary studies have shown that the intracellular concentrations of the polyamines is highly regulated at many steps in their biosynthesis, catabolism and transport. The fact that cells contain such complex apparatus for the tight control of the levels of these molecules shows that only a very narrow concentration range is tolerated.
Polyamine transport into mammalian cells is energy and temperature dependent, saturable, carrier-mediated and operates against a substantial concentration gradient (Seiler, N. et al. Polyamine transport in mammalian cells. Int. J. Biochem. 1990, 22, 211-218; Khan, N. A.; Quemener, V. et al. Characterization of polyamine transport pathways, in Neuropharmacology of Polyamines (Carter, C., ed)., 1994, Academic, San Diego, pp. 37-60). Ample experimental evidence exists that polyamine concentration homeostasis is mediated via this transport system. Changes in the requirements for polyamines in response to growth stimulation is reflected by increases in the transport activity. Stimulation of human fibroblasts to cell proliferation by serum or epidermal growth factor was followed by an 18-100 fold increase in the uptake of putrescine (Dipasquale, A. et al. Epidermal growth factor stimulates putrescine transport and ornithine decarboxylase activity in cultures human fibroblasts. Exp. Cell Res. 1978, 116, 317-323; Pohjanpelto, P. Putrescine transport is greatly increased in human fibroblasts initiated to proliferate. J. Cell Biol. 1976, 68, 512-520). Tumors have been shown to have an increased rate of putrescine uptake (Volkow, N. et al. Labeled putrescine as a probe in brain tumors. Science, 1983, 221, 673-675; Moulinoux, J-P. et al. Biological significance of circulating polyamines in oncology. Cell. Mol. Biol. 1991, 37, 773-783).
Inhibition of polyamine biosynthesis in cells in culture by α-difluoromethylomithine (DFMO), a well-studied mechanism-based inhibitor of ODC, causes a substantial depletion of intracellular putrescine and spermidine with resultant cell growth inhibition. Upon supplementing the culture media with exogenous polyamines this depletion causes transport activity to rise several-fold (Bogle, R. G. et al. Endothelial polyamine uptake: selective stimulation by L-arginine deprivation or polyamine depletion. Am. J. Physiol. 1994, 266, C776-C783; Alhonen-Hongisto, L. et al. Intracellular putrescine deprivation induces uptake of the natural polyamines and methylglyoxal bis(guanylhydrozone). Biochem. J. 1980, 192, 941-945). The cells then returned to their original rate of growth.
Genes for the polyamine transport protein or complex have been cloned from Escherichia coli and yeast (Kashiwagi, K. et al. J. Biol. Chem. 1990, 265, 20893-20897; Tomitori, H. et al. Identification of a gene for a polyamine transport protein in yeast. J. Biol. Chem. 1999, 274, 3265-3267). The genes for the mammalian transporter await identification. A subunit of the transporter from E. coli has been crystallized and its X-ray structure has been determined (Sugiyama, S. et al. Crystal structure of PotD, the primary receptor of the polyamine transport system in Escherichia Coli. J. Biol. Chem. 1996, 271, 9519-9525). This structure represents one of a few but growing number solved for spermidine-binding proteins. Since this structure was determined on a prokaryotic species its use in the design of mammalian transport inhibitors was deemed to be of limited value.
Several researchers have studied the ability of polyamine analogs to inhibit the uptake of 3H-spermidine into cells. Bergeron and coworkers studied the effect of addition of different alkyl group substitutions on the terminal nitrogen atoms of spermidine or spermine analogs (Bergeron, R. J. et al. Antiproliferative properties of polyamine analogs: a structure-activity study. J. Med. Chem. 1994, 37, 3464-3476). They showed that larger alkyl groups diminished the ability to prevent uptake of radiolabeled spermidine. They later concluded that increases in the number of methylenes between the nitrogen atoms decreased the ability to compete for 3H spermidine uptake (Bergeron, R. J. et al. A comparison of structure-activity relationships between spermidine and spermine antineoplastics. J. Med. Chem. 1997, 40, 1475-1494). They also concluded that the polyamine transport apparatus requires only three cationic centers for polyamine recognition and transport (Porter, C. W. et al. J. Cancer Res. 1984, 44, 126-128). Two groups have analyzed literature examples of the polyamine analogs' ability to inhibit 3H spermidine uptake into L1210 cells by CoMFA and QSAR methods (Li, Y. et al. Comparative molecular field analysis-based predictive model of structure-function relationships of polyamine transport inhibitors in L1210 cells. Cancer Res. 1997, 57, 234-239; Xia, C. Q. et al. QSAR analysis of polyamine transport inhibitors in L1210 cells. J. Drug Target. 1998, 6, 65-77).
A radiochemical assay is used for biochemical analysis of transport and has been used to study polyamine transport in yeast and a variety of mammalian cells (Kakinuma, Y. et al., Biochem. Biophys. Res. Comm. 216:985-992, 1995; Seiler, N. et al, Int. J. Biochem. Cell Biol. 28:843-861, 1996). See, for example Huber, M. et al. Cancer Res. 55:934-943, 1995.
Many undesirable skin conditions are defined by uncharacteristically proliferative cell growth of the underlying tissue. Clinical conditions range into various broad classes from the extremely fast growth and life-threatening malignant cancerous disease states to immunological disease states such as cutaneous lupus, atopic dermatitis and psoriasis. The inhibition of unwanted hair growth, although not a life-threatening disease state, is nevertheless a very important cosmetic problem. Any therapeutic or treatment that could reduce that growth of the skin tissue or hair would be useful in these conditions.
Mammalian hair growth is cyclic and is composed of anagen (hair growth), catagen (follicle regression) and telogen (resting) phases. In mice, ornithine decarboxylate (ODC) is expressed in ectodermal cells at sites where hair follicles develop during embryonic development (Nancarrow, M. J., et al., Mech. Dev. 84: 161-164 (1999); Schweier, J. In: Molecular Biology of the Skin: The Keratinocyte, Darmon M, et al., Eds., Academic Press, New York, 1993, pp 33-78).
In proliferating bulb cells of anagen follicles, ODC is abundantly expressed except for a pocket of cells at its base. It has been hypothesized that local mediators such as fibroblast growth factors or BMPs induce the expression of ODC in a transitory fashion resulting in the hair follicle to enter the anagen phase of growth (Soler, et al., Modulation of murine hair follicle function by alterations in ornithine decarboxylase activity. J. Invest. Dermatol. 1996, 106 (5), 1108-1113).
After the local concentration of these growth factors decline, ODC activity is also reduced allowing the follicles to enter catagen phase. ODC protein expression does not resume until new follicle growth cycle commences. A more complex expression of ODC is found in vibrissae (beard hair). ODC is expressed in the keratinocytes of the vibrissal hair shaft as well as in the bulb and outer root sheath cells near the follicle bulge. In comparison, ODC expression is very low in interfollicular epidermis. For a review of the biology of hair growth control, see Messenger, A. G. The control of hair growth: an overview. J. Invest. Dermatol., 1993, 101(1), 4S-9S. The polyamine concentrations in normal human epidermis have been measured. (El Baze et al., Distribution of polyamines in human epidermas. British J. Dermatology 1985, 112, 393-396).
It has been shown that polyamine levels, together with the levels of their biosynthetic enzymatic activities are elevated in proliferative states. (Cohen, S. S. A Guide to the polyamines, Oxford University Press, 1998, New York).
The first, dedicated and rate-limiting step in polyamine biosynthesis is the well-examined enzyme ornithine decarboxylase (ODC). Elevated levels of this enzyme in a variety of proliferating tissue types has led to the designation of its gene as a protooncogene. (Auvinen et al., Ornithine decarboxylase activity is critical for cell transformation. Nature, 1992, 360, 355-358). Despite its consistent appearance in transformed tissues, its overexpression is not in itself sufficient for tumor formation. (Clifford, A et al., Cancer Res. 1995, 55, 1680-1686).
An initiation event, either chemical or physical, appears to be required for transformation to occur. An example of a physical initiation event and the resulting increase in levels of skin polyamines can be found in the work of Seiler and Knodgen. (Seiler et al., Effects of ultraviolet light on epidermal polyamine metabolism. Biochemical Med., 1979, 21, 168-181). Following a 10 minute UV exposure, the concentrations of skin polyamines, putrescine and spermidine, were shown to be elevated by 5-fold and 2-fold respectively.
O'Brien and coworkers explored the effect of a variety of chemical inducing agents including 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the skin levels of ODC activity. (O'Brien et al., Induction of the polyamine-biosynthetic enzymes in mouse epidermis by tumor-promoting agents. Cancer Res. 1975, 35(7), 1662-70). The activity reached a peak (230-fold greater than control) at 4 to 5 hr after TPA treatment and returned to control level by 12 hr. Numerous other studies showed that treatment of skin with chemical or physically insults results in an increase in the enzymatic activity of ODC with a corresponding increase in the levels of the polyamines. (Scalabrino et al., Levels of activity of the polyamine biosynthetic decarboxylases as indicators of degree of malignancy of human cutaneous epitheliomas. J. Invest Dermatol. 1980, 74(3), 122-4; O'Brien T G. The induction of ornithine decarboxylase as an early, possibly obligatory, event in mouse skin carcinogenesis. Cancer Res. 1976, 36(7 PT 2), 2644-53; Young et al., UV wavelength dependence for the induction of ornithine decarboxylate activity in hairless mouse epidermis. Carcinogenesis, 1986, 7(4), 601-604).
It has been hypothesized that increases in ODC activity associated with skin is almost entirely due to high cellular turnover time of hair follicles (18-23 h) and hence a reflection of their highly proliferative state. (Hynd, P. I. et al., Inhibition of polyamine synthesis alters hair follicle function and fiber composition. J. Invest. Dermtol. 1996, 106(2), 249-253).
In a study by Hynd and coworkers, sheep were treated systemically with DFMO and minimal effects on fiber growth (10% decline), fiber diameter (14% increase) and sulfur content (increased) of hair were measured. Detailed experiments into the effects of DFMO treatment on the protein composition of fibers and levels of keratin gene mRNA were performed in an attempt to explain the unexpected lack of effects on growth. These authors also used a cultured follicle fiber growth assay to explore other treatment options. The inclusion of 500 μM DFMO, a well studied inhibitor of ODC, to the culture media had no effect on the growth of cultured follicles, paralleling the result on the animal. It was found that treatment with the AdoMet decarboxylase inhibitor MGBG caused potent (ED50˜5 μM) inhibition of the growth of cultured follicles. This growth inhibition was paralleled by the effects of MGBG on DNA synthesis as measured by a tritiated thymidine incorporation assay.
An experiment in strong support of the requirement of spermidine for hair growth was the addition of spermidine (50 μM) to the MGBG (10 mM) treated cultured hair follicles. Complete recovery of DNA synthesis was observed. Addition of spermine (50 μM) did not overcome the MGBG-mediated inhibition. This would seem to suggest the important role played by spermidine in hair growth. Furthermore, the use of a specific spermine synthase inhibitor (N-butyl-1,3-diaminopropane (50 μM)) had no effect on the growth of the cultured follicles. This study suggests that prior experiments associating ODC activity to hair growth were mistakenly directed towards that enzyme's product, putrescine and not the higher polyamines, especially spermidine. The authors of this paper state that “This suggests that spermidine is the essential polyamine for normal fiber growth . . . ”
The importance of a specific individual polyamine to cellular growth and metabolism has been debated routinely in the scientific literature. Discussed herein below, it has been found to be very difficult to specifically perturb individual polyamines in an experimental setting. Compensatory mechanisms give rise to adjustments elsewhere in the pathway allowing the system to overcome whatever block has been imposed. An important modem tool has recently been brought to bear on this problem. Through the transgenic introduction of the rate-limiting catabolic enzyme responsible for the elimination of polyamine, spermidine/spermine acetyltransferase (SSAT), a marked change of the polyamine pools was noted. (Pietila et al., Activation of polyamine catabolism profoundly alters tissue polyamine pools and affects hair growth and female fertility in transgenic mice overexpressing spermidine/spermine N1-acetyltransferase. J. Biol. Chem. 1997, 272(30), 1876-51). Dramatic increases in the levels of putrescine together with increased enzymatic levels of S-adenosylmethioninedecarboxylase (AdoMetDC) were observed in the skin of transgenic mice. Only slight changes in the levels of the other polyamines, spermidine and spermine, were noted. The increased putrescine levels were explained by the metabolic disassembly of the higher polyamines via the introduced enzyme SSAT. This introduction of SSAT into these transgenic animals had a profound effect on their hair growth. Normal hair follicles were replaced with large cysts filled with a keratin-like substance. The epidermis of the transgenics was thickened and no hair shafts were seen. Pietila et al concluded that the increased level of putrescine in the skin of these animals led to the profound phenotypical changes observed.
An additional transgenic study supports the hypothesis that perturbations in the controls on putrescine levels have a dramatic effect on hair growth control. In 1996 O'Brien and coworkers reported on their studies on the transgenic introduction of gene for mutated ODC specifically associated with skin into mice. (Soler et al., supra). The transgene used in these studies used a skin-specific keratin 6 promoter. This study measured increased levels of putrescine in the skin of transgenic mice with little effect on the levels of spermidine or spermine. Follicular cysts with no hair growth were noted in these animals. DFMO given at 1% in the drinking water prevented this loss of hair growth when given before the first hair cycle had started. Furthermore, DFMO given after the above loss of hair caused the partial restoration of hair growth and completely regenerated normal follicle appearance. Soler et al. concluded that intracellular putrescine acts as a molecular switch regulating the behavior of the keratinocytes of the hair follicle. These studies were followed by an additional study using double transgenic mice over-expressing both SSAT and ODC. (Pietila et al., Relation of skin polyamines to hairless phenotype in transgenic mice overexpressing spermidine/spermine N1-acetyltransferase. J. Invest. Dermatol. 2001, 116(5), 801-805). Similar phenotypical changes were observed in this case also. In all of these studies the introduction of the SSAT or ODC transgene or both, despite paradoxical expectations to decrease or increase total polyamine levels respectively, resulted in elevated levels of putrescine. The resulting common phenotype of lack of hair growth and keratin filled cysts points to the importance of putrescine in skin biology.
Weekes et al showed that putrescine and its chain-extended analogs act as potent inhibitors of ODC induction when applied topically to mice prior to TPA treatment. (Weekes et al., Inhibition by putrescine of the induction of epidermal ornithine decarboxylase activity and tumor promotion caused by 12-O-tetradecanoylphorbol-13-acetate. Cancer Res., 1980, 40, 4013-4018). They showed that the topical application of 20 μmole of putrescine 2 hr after the TPA treatment inhibited the induction of ODC activity by 50%. Treament with spermidine, 1,7-diaminoheptane and spermine gave 90% inhibition when used at the same concentration. These same concentrations, when added directly to the assay medium, had no effect on the assay system. Furthermore, putrescine did not induce the production of detectable levels of ODC-antizyme in the mouse epidermis. Therefore, these diamines are inhibiting the induction of ODC by TPA. In the transgenic studies referenced above, the elevated putrescine produced maybe acting in a similar fashion, inhibiting the induction of ODC enzymatic activity.
It is now apparent that perturbations of cutaneous polyamine levels will affect hair growth. Numerous studies have shown that inhibition of ODC with α-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, reduces hair growth in mammals. Mice were found to have reduced hair growth when DFMO was systemically delivered via the drinking water (Takigawa, M. et. al., Cancer Res. 43:3732-3738 (1983)). Intravenous administration of DFMO decreased wool growth in sheep (Hynd, P. I. et. al., J. Invest. Dermatol. 106:249-253 (1996)) and oral administration of DFMO in cats and dogs produced alopecia and dermatitis (Crowell, J. A. et. al, Fundam. Appl. Toxicol. 22:341-354 (1994)). Additional evidence that ODC plays a role in hair follicle regulation resulted from a study in humans that were being treated for acute Trypanosoma brucei infections (African sleeping sickness) (Pepin, J. et. al. Lancet 2:1431-1433 (1987)) using DFMO. Patients using this treatment showed signs of hair loss mainly on the scalp but it was reversible after discontinuing treatment.
Polyamine biosynthesis has also been shown to be essential during the activation of immunocompetent cells (Fillingame, R. H. et. al., Proc.Natl.Acad.Sci. USA 72:4042-4045, (1975); Korpela, H. et. al., Biochem. J. 196:733-738 (1981)). Studies with DFMO confirm that polyamine depletion therapy can inhibit the immune response and may be a successful therapy against a number of autoimmune diseases. Both humoral and cell-mediated immune responses were affected by the anti-proliferative effect of polyamine depletion.
DFMO treatment of mice challenged with tumor allografts resulted in modified cytotoxic T-lymphocyte and antibody responses (Ehrke, J. M. et. al., Cancer Res. 46:2798-2803 (1986)). Reports by Singh et al. indicate that DFMO treatment may also ameliorate acute lethal graft versus host (ALGVH) disease in mice (Singh, A. B. et. al., Clin.Immunol. Immunopathol. 65:242-246 (1992)). Murine ALGVH represents a model of human GVH that contributes to the morbidity and mortality of bone marrow transplantation in humans and is characterized by anemia and the loss of T cell function and numbers. In this study, treatment of ALGVH mice with DFMO decreased mortality and anemia while preserving the cytotoxic T cell and natural killer cell population of the host.
Polyamine depletion therapy using DFMO has also been shown to benefit lupus-prone female NZB/W mice (Thomas, T. J. et. al., J.Rheumatol. 18:215-222 (1991)). Anti-DNA antibody production, immunoglobulin G and A synthesis, proteinuria and blood urea nitrogen were significantly reduced in treated mice. These studies indicate that polyamine depletion may beneficially treat several clinical autoimmune diseases including host versus graft disease, graft versus host disease and lupus. This antiproliferative strategy could be applied to other autoimmune diseases including cutaneous lupus, multiple sclerosis, atopic dermatitis, rheumatoid arthritis, scleroderma, inflammatory bowel disease, transplantation rejection and diabetes.
In modern societies the basic biological function of human hair, protection from the environment, has been lost. It is now desired that hair be removed from many parts of the human body in order to give a more cosmetically pleasing and socially acceptable appearance. There are many currently used methods to remove unwanted hair, none of which is entirely acceptable. These methods include; shaving, electrolysis, depilatory creams or lotions, waxing, tape-striping, depilatory devices, laser-mediated removal, tweezers plucking. Additionally, there are many examples of less than effective chemical hair growth inhibitors. Many of these currently used methods cause the undesired effect of thicker hair re-growth.
Examples of agents used in skin care products that have the effect of hair growth inhibition include agmatine, BHT or BHA, cetyl pyridinium chloride, bexamidine, ursolic acid, green tea catechins, phytosterols. Other agents known in the art to inhibit hair growth include: 1,10-phenanthroline; 5′-para-fluorosulphonyl benzoyl adenosine; 5-keto-D-fructose; 5-keto-D-fructose-1,6-bisphosphate; 6-amino-6-deoxy-glucose; agaric acid, 8-bromo-cAMP; cysteine sulphinic acid, D-mannosamine; diethylaminomalonate; doxycycline; ethacrynic acid; ethoxyquin; eupacunin; euparoin acetate; fluvastatin; guanidinosuccinic acid; inhibitor of a cysteine pathway enzyme; methacycline; mevastatin; mevinolin; minocycline; N-alpha-(p-tosyl)-L-lysine chloromethyl ketone; N-acetyl-beta-D-mannosoamine; oxaloacetic acid; phosphoclycerate; pravastatin; protocatechuic aldehyde; quinaldic acid; rivastatin; simvastatin; squalestatin; taxodione; taxodone; tetracycline; vemolepin; 1,8-diaminooctane; 2-methyl-6-heptyne-2,5-diamine; 3-carboxpropyl disulphide; saw palmetto extract; willow herb extract; pumpkin seed extract; 5-(N-benzyloxycarboyl-1-phenylalanamidomethyl)-3-bromo-4,5-dihydroisoxazole; 5′-deoxy-5′-methylthioadenosine; 6-heptyne-2,4-diamine; actinonin; alpha-methyl-DL-methionine; alpha-ethyl-ornithine; apigenin; arginase inhibitor; batimistat; caffeic acid; captopril; chlorotaurine; cholesterol pathway enzyme inhibitor; cyclooxygenase inhibitor; cysteamine; cysteinyl-glycine; d-cysteine; D-penicillamine; difluoromethylornithine (DFMO); L-difluoromethylornithine (L-DFMO); diethyl glyoxalbis(quanylhydrazone); diethyldithiocarbamic acid; dimethylcysteamine; doxycycline; eicosapentaenoic acid; estramustine; ethacrynic acid; etoposide; H-homoarginine; inhibitor of the formation of glycoproteins; proteoglycans or glycosaminoglycans; inhibitor of hypusine biosynthetic pathway; L-alanosine; L-argininamide; L-asparaginamide; L-cysteine methyl ester; lipoic acid; lovastatin; marimistat; matlystatin-B; meso-dimercaptosuccinic acid; methacycline; methylglyoxal bis(guanylhydrazone); minocycline; N(gamma)-methyl-L-arginine; N-[N[((R)-1-phosphonopropyl)-(S)-leucyl]-(S)-phenylalanine-N-methylamide; N-acetylcysteine; N-phosphonalkyl dipeptides; N-phosphonoacetyl-aspartic acid; nalidixic acid; N-alpha-acetyl-L-arginine methyl ester; N-gamma-L-arginine benzyl ester; N-gamma-nitro-L-arginine; N-gamma-nitro-L-arginine methyl ester; nordihydroguaianetic acid (NDGA); extract from creosote; oxaloacetic acid; pantothenic acid; pantothenic acid analogs; phosphocysteamine; propyl gallate; protein kinase C inhibitor; quercetin; S-carbamyl-L-cysteine; S-trityl-L-cysteine; sulphasalazine; suppressor of angiogenesis; tetracycline; thiosalicyclic acid; tyramine; herbimycin; HNMPA(AM)3; inhibitor of alkaline phosphatase; lavendustin A; methyl caffeate; protein-tyrosine kinase inhibitors; tryphosstin A47; O-p-nitrohydroxylamine; alpha-fluoromethylhistidine; mycophenolic acid; bromocryptine; cromoglycate; quinoline-3-carboxamide; 16 alpha or beta-substituted 4-aza-5-alpha-androst-1-en-3-ones; 2-aryl-indole derivatives; 2-phenyl-3-aminoalkyl-indole derivatives; 3-oxo-4-aza-5-alpha-androstane derivatives; 5-alpha-androstan-3-ones; 5-(aminocarbonylalkyl)-3-heterobicyclyl-alkylaminoalkyl)-2-phenylindole derivatives; 6-azaindole derivatives; 7-azaindole derivatives; aryl-imidazo-pyridines; finasteride; GnRH inhibitors; aloe; carboxylalkylamine derivatives; clove; Echinacea angustifolia; Echinacea purpurea; elasatin decomposition enzyme inhibitor; extracts from ginger; hydrolyzing almond; lithosperumum; peptides; extract of rosaceae; extract of sanguiosorba officinalis; tropaeolum majus; extract of white birch and rubiaceae plant groups; extract of juniperus genus and/or malt extract; malonamide derivatives; elastase inhibitor; papain; trypsin; chymotrypsin; pepsin; bromelain; ficin or pancreatin; plant fruit enzyme extracts; compounds from pleinoe sp; curcuma longa L or Biopyros kaki; 2-indole carboxylic acid derivatives; alpha-TNF antagonist; aminopropanes; bacterium ribosomes; non-steroidal anti-inflammatory drugs (NSAIDS); diethylenediamines; histamine antagonist; interleukin-1 antagonist; lipoxygenase inhibitors and stimulants; phenothizaines; sulfotransferase inhibitors; tetrazolyl-benzofuran carboxamides; tetraazolylbenzothiophene carboxamides; cyanoguanidine derivatives; 17alpha-hydroxy-4,9(11)-pregnadiene-3,20-dione derivatives; anti-angiogenic steroids; pyrimidine-cyanoguanidine derivatives; substituted amidine or guanidine; benzothotphene derivatives; (−)-cis-6(S)-phenyl-5(R)-[4-(2-pyrrolidin-1-ylethoxyphenyl]-5,6,7,8-tetrahydronaphehenen-2-ol D-tartrate ietrahydronaphthalene derivatives; estrogen agonists or antagonists; tetrahydroisoquinolines; heptapeptide luteinising hormone releasing hormone (LHRH) analogs; 3-(anilinomethylene)oxindole derivatives; bezo-[f]-quinolin-3-one derivative; ((S-(−)-N-(alpha-ethylbenzyl)-3-hydroxy-2-phenylquinoline-4-carboxamide); 24-ethyl-(delta)-4,22-cholestadien-3-one; extracts from Hydrangea macrophylla; Hydrangea serrata; Iridaceae belamcanda adans; iridaceae Iris I; moraceae humulus; CDK binding proteins; chimeric polypeptide with cyclin-dependant binase (DK) binding motif; E6AP-binding polypeptides; 2-mercaptoethanol; 2-mercaptopropionic acid; cysteine; diethyldithiocarbamic acid; dithiothreitol; glutathione; homocysteine; lipoic acid; 3-mercaptopropionic acid; N-acetyl-L-cysteine; thiodiclycol; thiodicyclolic acid; thioglycerol; thioglycolic acid; thiolactic acid; thiomalic acid; thiosalicyclic acid; thioxanthine; benzoid acid lactone ether; 1-halomethyl-5-alpha-androstanes and delta-androstanes; hedgehog antagonists; patched antagonists; copper; iron; zinc; 1-dehydromelengestrol acetate; 1-dehydromegestrol acetate; chlormadinone acetate; cyproterone acetate; medroxyprogesterone acetate; megestrol acetate; melengestrol acetate; nomegestrol acetate; non-elastomeric polyolefin resin; partially fluorinated polyolefin resin; nucleic acid molecule; trypsin analogs; bacteriostatic or maemostyptic agnt; stannous fluoride; alpha- or gamma-linolenic acid; EGF; lipoxygenases; extract of regulo plant (abelmoschus moschatus); extract of wolo plant (borassus flabellifer); androstenedione analogs; activin A (polypeptide); hydrindanes; butyric acid derivatives; phytoestrogen; tetrahydroisoquinolines; tetrahydronaphthalenes; 3-amino-2,3-dihydrobenzoic acid; 6-fluoro-2,5-diamino hexanoic acid; (S)-2-amino-4-aminooxybutyric acid; extracts of fruits and other plant parts from serenoa repens; carrot oil; clove oil; diazo compounds; essential oil; honey; juniper oil; lavender oil; lemon juice; palmarosa oil; rosemary oil; sugar; sugary substance; thuja oil; triarylmethane compounds; S-nitrosoglutathione; 1-diethyl-2-hydroxy-2-nitrosohydrazine; S-nitrosocysteine; nitroglycerin; perfluoro-substituted aniline derivatives; 17-alpha-propyltestosterone; 4-androstene-3-one-17-beta-carboxylic acid; (4R)-5,10-seco-19-norpregna-4,5-diene-3,10,20-trione; chlormadinone acetate; cyproterone acetate; progesterone; spironolactone; melatonin; Boeman Birk inhibitor (soy derived); 2-substituted-6-tetrahydronaphthyl or indanyl naphthalene derivatives; aqueous alcoholic extract from genus Centipeda; antaonist of hedgehog signal transduction pathway; epidermal growth factor (EGF); finasteride; fatty acids; 2-phenylbenzothiophene derivatives; 2-arylimino-oxaza or thiaza heterocyclic compounds; glutathione synthesis stimulators; indole derivatives; Cinnamonum verum; Curcurbita pepo; Epilobium roseum; Salvia officinalis; Serenoa repens; Cassia obtusifoila linne; polynucleotide; dormant cell extracts; N-substituted benzyl or thienylmethyl-4-pyridone compounds; (1H)-benzo(c)quinolizin-3-one derivatives; 5-alpha reductase inhibitors; adenylsuccinate synthetase inhibitors; aspartate transcarbamylase inhibitor; gammaglutamyl traspeptidase inhibitor; ornithine decarboxylase inhibitors; citric acid; Dead Sea salt; vitamin K; Cucurbitaceae; extract of Ikurinin; phosphodiesterase inhibitors; phlondrin; phloretin; 5-alphaandrostene-3-alpha-17-betadiol; cyproterone; Hedera helix; Lithospermum root; medoroxyprogesterone; mestanolone; norethisterone; Scutellaria root; tomato; extract of Commiphora myrrha; Cymbopogon nardus; Lagerstroemia speciosa; phyllanthus nuriri; Smilaz zeylanica; Woodfordia fruticosa; chelating agents; chlorophenol; ortho-phenyl-phenol; niphtolide; peach oil; sorbic acid; Cistanche salso; Plantago asiatica; Stachys sieboldii; 2-amino-5-substituted benzophenone; aniline derivatives; bur marigold infusion; camphoroil; citric acid; conifer extract; daisy infusion; honey; sea-buckthom oil; tannin solution; capsicum; capsicum extract (Solanaceae family); conjucate comprising active agent substituted with amino acid peptide or trisamine carrying fatty acid ester and dithioalkanoyl groups; recombinant DNA encoding EGF; recombinant DNA encoding TGFalpha; 11-beta-aryl-17-spiro-pyrrolin-2-ylidene N-oxide steroid progestins and antiprogestins; phenyl imidazolidines; ammonium salt of weaker acids; essential oils; vitamin F; coumarin derivatives; leuteinizing hormone-releasing hormone; leuteinizing hormone-releasing hormone analogs; hydroxamic acid derivatives; bomeol; cineole; linalool; methyl heptenone; oil of ginger; shogaol; zingerone; zingiberol; zingiberone; glutathione S-transferase modulator; aminoacid(s); lipoxydase; inhibitor of glutamine metabolism thiomolybdate compound; hairless protein inhibitor; aromatase inhibitor; trifluoroanilide derivatives; EGF; EGF analogs; and extract of seeds of Coix lachrymal-jobi.
Shander et.al. describes the use of sulfhydryl active compounds as mammalian hair growth inhibitors. (U.S. Pat. No. 6,743,419).
Other categories of agents that may be added to a therapeutic or cosmetic product suitable for various cutaneous applications include: surfactants; desquamation actives; anti-acne agents; anti-wrinkle actives and anti-atrophy agents; vitamin B3 compounds; retinoids; anti-oxidant or radical scavenging agents; chelators, flavonoids; anti-inflammatory agents; anti-cellulite agents such as caffeine, theophylline, theobromine or aminophylline; tanning agents such as dihydroxyacetone; skin lightening agents such as kojic acid, arbutin, transexamic acid; antimicrobial agents useful for destroying disease or odor causing bacteria; sunscreening agents; and skin conditioning agents.
Numerous biochemical studies have shown that cellular polyamine concentrations are tightly regulated at various control points. Overall, these control points can be divided into those that control the biosynthesis of polyamines from amino acid derived precursor molecules, those that control the transport into the cell from external sources and finally those that degrade the higher polyamines into their smaller components. Of these three ways to obtain and interconvert between the various forms of the polyamines only the biosynthetic and transport pathways allow the cell to gain additional carbon atoms into the cycle.
Polyamine biosynthesis involves two highly regulated enzymes, ornithine decarboxylase and S-adenosylmethionine decarboxylase, and two constitutively expressed enzymes, spermidine synthase and spermine synthase. The activities of the first two enzymes, the decarboxylases, are tightly regulated through a variety of growth factors and by the presence of their products. The activities of the other two enzymes, the aminopropyltransferases (spermidine synthase and spermine synthase), are mainly controlled by the availability of their substrates (i.e. the products of the decarboxylase enzymes). Furthermore, the cell can compensate for its requirements for polyamines by a specific polyamine transport system. Increased needs for polyamines following stimulation with growth factors or decreased needs following over-incorporation of polyamines results in compensatory changes in transport activity. The biochemical rheostat or regulator by which the cell can control both polyamine biosynthesis and transport has been defined as the antizyme system.
Bey et al discussed mechanism-based irreversible inhibitors of polyamine biosynthesis of both the enzyme ornithine decarboxylose (ODC) substrate and product of the ODC enzyme. (Bey, P. et al., J. Med. Chem. 1983, 26, 1551-1556). A review of this research can be found in Bey, P. et al., in “Inhibition of Polyamine Metabolism” pp. 1-31 eds. P. P. McCann, A. E. Pegg and A. Sjoerdsma, Academic Press. 1987. In their review, Bey et al., categorize inhibitors of ODC into three major types: (1) analogs of substrate or product which act as competitive inhibitors; (2) molecules capable of interacting or combining with the PLP cofactor; and (3) enzyme-activated irreversible inhibitors designed on either the substrate or product.
A good deal of research has defined the requirements for inhibitors of analogs of ornithine and putrescine. The L-configuration of the α-amino acid is preferred for substrate analogs of ornithine. A potent reversible inhibitor, α-methylornithine 1, shows this profound stereochemical preference (L-(S)-α-methylornithine Ki=19 μM; D-(R)-α-methylornithine Ki=1300 μM). (Bey et al., J. Med. Chem. 1978, 21 (1), 50-55). The carboxyl group can be substituted by a tetrazoyl group. A distance of about 6 Å between the nitrogen atoms is believed to be optimal for binding to the active site. Structural features, such as unsaturation in a trans geometry, which favor this stretched conformation, increase the affinity to the enzyme. (Relyea et al., Biochem. Biophys. Res. Comm. 1975, 67, 392-402). Reylea et al. suggested that an adduct forms between 1,4-diamino-trans-2-butene 3 and the PLP cofactor before binding to the enzyme. The authors of the previous report (Rey et al) suggest that a hydrophobic interaction favors the binding of this molecule. Substitutions on the α-carbon are allowed (see α-methylomithine above). Substitutions on the δ-nitrogen of ornithine are not allowed.
Substrate based ODC inhibitors are shown below.                α-methylornithine, 1 (Ki=40 μM) (Bey et al, supra)        
                trans-1,4-diaminocyclohexane-1-carboxylic acid, 2 (Ki=70 μM) (Bey et al, supra)        
                1,4-Diamino-trans-2-butene, 3 (Ki=2 μM) (Reylea et al, supra)        
                trans-3-dehydroornithine (Ki=4.4 μM) (Reylea et al, supra)        
                1,4-Phenylenediamine 4 (Ki=46 μM) (Solano et al., The 1,2 and 1, 3 isomers of Phenylenediamine were essentially inactive; Int. J. Biochem. 1988, 20 (4), 463-470).        
                1,4-Dimethylputrescine (Moyano et al., J. Med. Chem. 1990, 33 (7), 1969-74        
                p-Aminomethyl-phenylglycine (Ben-ishai et al., Tetrahedron, 1977, 33, 2715-2717).        
                trans-1,4-diaminocyclohexane (CAS RN [2615-25-0])        
                trans-1,4-diaminomethylcyclohexane        
                Di-α-methyl-p-phenylenediamine        
                1,4-Diaminomethylnaphthylene        
                N,N′-Bis(3-aminopropyl)-2-butene-1,4-diamine (CAS RN [110319-68-1])        

The x-ray crystal structure of ODC has recently been determined. (Almrud et al., J. Mol. Biol. 2000, 295(1), 7-16). A generally accepted assumption in the polyamine field is that it requires an irreversible ODC inhibitor to effectively deplete cellular polyamines. Explanations for this include the fast turnover of the enzyme, lack of specificity and the general lack of potency of competitive inhibitors. Furthermore, several of these have been shown to increase the apparent half-life of the enzyme through stabilization. (Harik et al., Mol. Pharmacol. 1974, 10, 41-47; McCann et al., Biochem. Biophys. Res. Comm. 1977, 76, 893-899).
Cofactor interaction-based inhibitors exploit the requirement in all α-amino acid decarboxylase enzymes for the vitamin B6 cofactor pyridoxal phosphate (PLP). By incorporating a strongly nucleophilic moiety into a substrate or product analog an extremely stable adduct is formed between PLP and the inhibitor. Several of these molecules are extremely potent inhibitors with Ki values into the low nanomolar range. α-Hydrazinoornithine showed a Ki value of 0.5 μM. (Inoue et al., J. Biochem (Tokyo), 1975, 77, 879-893). An improved method of synthesis of this molecule has been reported. (Sawayama et al., Chem Pharm. Bull. 1976, 24, 326-329). A set of aminooxy putrescine analogs have been described which are extremely potent. (Khomutov et al., Biochem Biophys Res. Commun. 1985, 130, 596-602). The second of these shown below, 1-aminooxy-3-aminobutane, has a methyl group in the α-position to the amino functionality to prevent oxidative metabolism by amine oxidase enzymes.                α-Hydrazinoomithine (Ki=0.5 μM) (Kahana et al., Proc Natl Acad Sci USA 1984, 81(12), 3645-9).        
                1-Aminooxy-3-aminopropane (Ki=0.0032 μM) (Sawayama et al., Chem Pharm. Bull. 1976, 24,326-329).        
                1-Aminooxy-3-aminobutane (Ki=0.0028 μM) (U.S. Pat. No. 5,610,195 to Frei et al).        
                1-aminooxy-3-amino-2,2-difluoropropane        
                1-hyrazino-3-aminopropane        
                1-Aminooxy-3-amino-3-methylbutane        
                1,2-diaminooxyethane        

Because of microscopic reversibility of the protonation step in the mechanism of ODC, latent chemically reactive groups can be placed on either a substrate or product analog to produce a mechanism-based ODC inhibitor. (Bey et al., Inhibition of Polyamine Metabolism, supra). Tables 1 and 2 show various substrate analogs while Tables 3 and 4 show product analog inhibitors. In these tables Ki is the apparent dissociation constant and τ1/2 is the time of half-inactivation extrapolated to infinite concentration of inhibitor. Lower numbers in both instances mean better inhibitors. Data in the tables are from rat liver enzyme.
Introduction of a double bond in the β,λ-position of these analogs generated analogs with much higher activity. The difluoromethyl analogs in both the saturated (DFMO) and unsaturated analogs have been resolved into their enatiomerically pure forms. The (−)-versions are more active.
TABLE 1Reaction catalyzed by ODC and substrate analog mechanism-basedinhibitors. R GroupKi value (μM)τ50 (min)Reference—CHF2393.1Metcalf et al.(DFMO)J.Am. Chem.Soc. 1978, 100,2551-2553—CH2ClNo satd kineticst1/2 = 22 minSawayama et. al,at 100 μMsupra—CH2F751.6Metcalf et al.J.Am. Chem.Soc. 1978, 100,2551-2553—CH2CN870029Sawayama et. al,supra—CH═CH281027Danzin et al. J.Med. Chem.1981, 24, 16-20—CCH108.5Metcalf et al.J.Am. Chem.Soc. 1978, 100,2551-2553—CH═C═CH2??Castelhano, etal., J. Chem.Soc. 1984, 106,2734-2735
TABLE 2Unsaturated substrate mechanism-based ODC inhibitors. R GroupKi value (μM)τ50 (min)Reference—CH32.7Not a mechanism-U.S. Pat. No. 5,610,195based inhibitor—CH2F2.72.6Metcalf et al, supra—CHF2302.6U.S. Pat. No. 5,610,195
In terms of the latent reactive functionality, the product analogs shown in Table 3 generally follow the same trends in potency as the substrate analogs in Tables 1 and 2. An unfortunate coincidence occurs with α-ethynylputrescine and α-fluoromethyl-putrescine when used in vivo. These analogs inhibit not only ODC but also the enzyme 4-aminobutyrate transaminase (GABA-transaminase). Since these analogs are substrates for monoamine oxidase they are converted to the GABA analogs, which potently inhibit the GABA-transaminase. This is not acceptable due to the profound CNS effects of such inhibitors.
Introduction of a α-methyl group is known to prevent oxidation by monoamine oxidase enzymes and substituents are allowed on the δ-position of ODC substrates thus addition of such a methyl group was successfully tried. This analog, δ-methyl-α-ethynylputrescine, was resistant to oxidation and retained high ODC inhibitory properties. Introduction of this methyl group presents a substantial stereochemical problem since there are now 4 possible diastereoisomers. Each was synthesized and the active molecule was found to be the (2R, 5R) isomer. (Casara et al., J. Chem. Soc. Perkin Trans. I, 1985, 2201-2207).
Subsequent work utilized an alternative chemical approach to prevention of the monoamine oxidase metabolism. Introduction of two fluorine atoms in the β-position of α-ethynylputrescine provides a molecule without the stereochemical liability of the α-methyl derivative while also preventing the oxidation problem. (Kendrick et al., J. Med. Chem., 1989, 32, 170-173). This molecule, 2,2-difluoro-5-hexyne-1,4-diamine, is shown in Table 3 (Ki=10 μM). In vivo data in rats using this inhibitor described in this paper shows a dramatically long-lasting dose-dependant inhibition of ODC of the ventral prostate tissue following oral administration (50 mg/kg by gavage still showed >50% inhibition after 24 h).
TABLE 3Product analog mechanism-based inhibitors of ODC. R GroupKi value (μM)τ50 (min)Reference—CH3520No time-dependantDanzin et al., Biochem.inhibitionPharmacol. 1982, 31, 3871-3878—CH2F564.4Casara et al, supra—CHF2307.4Casara et al, supraCH═CH254010Metcalf et al, supra—CCH2.39.7Sawayama et al, supra—CCHw/δ-CH313.51.8Danzin et al., J. Biochem.Biophys. Res. Comm. 1983,116, 237-243.—CCHw/γ-CF2102.4Castechano et al, supra.—CH═C═CH21608Danzin et al, FEBS Lett.,1984, 174, 275-278.
TABLE 4Unsaturated product analog mechanism-based inhibitors of ODC. R GroupKi value (μM)τ50 (min)Reference—CH2F420.2Bey et al., J. Med Chem,supra.—CHF2600.7Bey et al., Inhibition ofPolyamine Metabolism, supra.—CCH15Sawayama et al.,supra.d. Nitrosylation—The ODC enzyme protein has been shown to be nitrosylated resulting in inhibition of its activity. (Bauer et al., Nitric Oxide Inhibits Ornithine Decarboxylase via S-Nitrosylation of Cysteine 360 in the Active Site of the Enzyme. J. Biol. Chem., 2001, 276, 34458-34464).
Other ODC inhibitors described include retinoic acid. (Zheng et al., Regulation of the induction of ornithine decarboxylase in keratinocytes by retinoids. Biochem J. 1995, 309, 159-65). The following agents were discovered through the use of an in vitro cell-based assay which inhibited the TPA-mediated increase in ODC activity in cultured mouse epidermal 308 (ME 308) cells: apigenin, benzylisothiocyanate, curcumin, diallyl disulfide, N-(4-hydroxyphenyl)retinamide (4-HPR), menadione, miconazole, nordihydroguaiaretic acid (NDGA) and phenethyl isothiocyanate. (Lee et al., Evaluation of the potential of cancer chemopreventive activity mediated by inhibition of 12-O-tetradecanoyl phorbol 13-acetate-induced ornithine decarboxylase activity. Arch Pharm Res. 1999, 22(6), 559-64).
There also exists a potential biological method to inhibit the induction of skin ODC activity caused by a variety of chemical or physical treatments. A number of studies have explored the ability of various agents to prevent the physical (UV radiation) or chemical (12-O-tetradecanoylphorbol-13-acetate (TPA)) induced increase in ODC activity in the skin. In 1986, Black and coworkers explored the mode of action of butylated hydroxytoluene-mediated photoprotection. (Koone et al., A mode of action for butylated hydroxytoluene-mediated photoprotection J. Investig. Derm. 1986, 87 (3), 343-347). They used orally ingested BHT in a murine model for protection from photocarcinogenesis. They noted that BHT significantly decreases UV radiation induction of epidermal ornithine decarboxylase activity. This paper examines the possibility that BHT provides this ODC inhibition and the resulting carcinogenesis protection by altering the chemical or physical properties of the stratum corneum. Mice were treated orally (in food) with 0.5% BHT for two weeks prior to ODC activity determination. The control group did not received any BHT. A 65% more UV radiation transmission in these controls was observed after excising stratum corneum samples. BHT caused a 70% inhibition of measured ODC activity. No differences in ODC activity were observed in comparisons between BHT-fed animals that had their stratum corneum stripped away prior to UV irradiation and those of UV irradiated non-stripped controls. Black et al suggested that irritation by the stripping could not account for the induction of ODC. Furthermore, it was shown that transmission differences could not be attributed to changes in the thickness or number of layers of stratum corneum. Black et al suggested that the protective role induced by BHT is due to its effects on the chemical not physical properties of the stratum corneum, possibly through an antioxidant effect.
Kozumbo and coworkers explored the ability of various antioxidant compounds to prevent TPA-induced epidermal ODC activity. (Kozumbo et al., Inhibition by 2(3)-tert-butyl-4-hydroxyanisole and other antioxidants of epidermal ornithine decarboxylase activity induced by 12-O-tetradecanoylphorbol-13-acetate. Cancer Res. 1983, 43, 2555-2559). They monitored the inhibition of the induction of ODC activity resulting following TPA treatment. Various antioxidant agents including BHA and 14 various analogs were tested by topical application to mice. A structure-activity study was performed that implied that hydroxyl and tert-butyl substituents played a significant role in their antioxidant-mediated antagonism of the induction of ODC by TPA. Their free radical scavenging mechanism of action is supported by the experiment showing no ODC inhibition when the agent is placed in the assay medium.
Another report described the inhibitory effects of anti-inflammatory drugs, a steroid triamcinolone and a NSAID indomethacin, on the UV-based induction of ODC activity in mouse skin. (Lowe et al., Antiinflammatory drug effects on ultraviolet light-induced epidermal ornithine decarboxylase and DNA synthesis. J. Investig. Dermatol., 1980, 74, 418-420). Young and coworkers explored the wavelength dependence for the induction of ODC in the skin of UV irradiated mice. The results that this induction was highest between the 260 and 310 nm suggested that the chromophore was a protein. (Young et al., UV wavelength dependence for the induction of ornithine decarboxylate activity in hairless mouse epidermis. Carcinogenesis, 1986, 7(4), 601-604).
Work described by Shirahata showed that the design of specific and potent (<1 μM Ki) inhibitors of the aminopropyltransferase enzymes spermidine and spermine synthase is possible. (Shirahata et al., Putrescine or spermidine binding site of aminopropyltransferases and competitive inhibitors. Biochem. Pharm. 1991, 41(2), 205-212). The molecules described have the advantage of being small and simple to produce. They have outstanding appeal for use in dermal applications such as hair growth inhibition. Analogs for inhibition of spermidine synthase include (IC50 in μM): cyclohexylamine (8.1), trans-4-methylcyclohexylamine (1.7) and exo-2-aminonorbornane (5.5). Furthermore, the simple molecules 5-amino-1-pentene, 1-butylamine (3.8) and 1-pentylamine (3.6) compounds showed respectable activity.
The effect of treatment with spermidine and spermine synthase inhibitors on the levels of polyamines in rat tissues is described in Shirahata et al., Effects of inhibitors of spermidine sythase and spermine synthase on polyamine synthesis in rat tissues. Biochem Pharmacol. 1993, 45(9), 1897-903. Oral administration of 4-methylcyclohexylamine (4MCHA) or N-(3-aminopropyl)cyclohexylamine (APCHA) at 0.02% or 0.1% in drinking water resulted in significantly reduced levels of spermidine or spermine in rat prostate. Lesser reductions were observed in rat liver, kidney or brain. Only very small amounts of drugs were seen in these tissues following this delivery method. A note was made to the relatively low toxicities of these two compounds. The LD50 values in rats are more than 250 mg/kg for 4MCHA and 500 mg/kg for APCHA for a 10-day treatment with drug in drinking water. A further point made by the paper was the inability of spermine depletion alone to affect the weight of the tissues.
Baillon et al describe the effect of some simple analogs of diaminopropane against spermine synthase using both isolated enzyme and whole cells. (Baillon et al., Inhibition of mammalian spermine synthase by N-aklylated-1,3-diaminopropane derivatives in vitro and in cultured rat hepatoma cells. Euro. J. Biochem. 1989, 179, 17-21). Potent inhibition of the enzyme was noted in both settings. Treatment of cells resulted in the appearance of drug in the cell (N-butyl-1,3-diaminopropane was the best with a Ki of 11.9 nM) showing effective uptake of the molecule. No great effect was seen on cellular polyamine levels (spermine and putrescine were reduced while spermidine levels were higher) following treatment with 50 μM N-butyl-1,3-diaminopropane. Baillon et al confirmed the potent activity of n-butylamine against the enzyme spermidine synthase (Ki=0.52 μM). They conclude with the statement, “altogether these findings are consistent with the idea that spermine synthesis is not needed for a normal growth rate provided that a compensatory increase in spermidine occurs.”
Other spermidine or spermine synthase inhibitors include the multisubstrate analog inhibitors reported by Coward and coworkers. These include adenosylspernidine AdoDATO, and AdoDATAD. (Lakanen et al., Synthesis and biochemical evaluation of adenosylspermidine, a nucleoside-polyamine adduct inhibitor of spermidine synthase. J. Med. Chem. 1995, 38(14), 2714-27; Tang et al., Synthesis and evaluation of some stable multisubstrate adducts as specific inhibitors of spermidine synthase. J. Med. Chem. 1981, 24(11), 1277-84; and Woster et al., Synthesis and biological evaluation of S-adenosyl-1, 12-diamino-3-thio-9-azadodecane, a multisubstrate adduct inhibitor of spermine synthase. J. Med. Chem. 1989, 32(6), 1300-7; respectively).
Polyamine transport into mammalian cells is energy and temperature dependent, saturable, carrier mediated and operates against a substantial concentration gradient. (Seiler, N. et al., Polyamine transport in mammalian cells. Int. J. Biochem. 1990, 22, 211-218; and Khan, et al., Characterization of polyamine transport pathways, in Neuropharmacology of Polyamines (Carter, C., ed.), 1994, Academic, San Diego, pp. 37-60). Ample experimental evidence exists that polyamine concentration homeostasis is mediated via this transport system. Changes in the requirements for polyamines in response to growth stimulation are reflected by increases in the transport activity. Stimulation of human fibroblasts to cell proliferation by serum or epidermal growth factor was followed by an 18-100 fold increase in the uptake of putrescine (DiPasquale, A. et al., Epidermal growth factor stimulates putrescine transport and ornithine decarboxylase activity in cultures human fibroblasts. Exp. Cell Res. 1978, 116, 317-323; and Pohjanpelto, P. Putrescine transport is greatly increased in human fibroblasts initiated to proliferate. J. Cell Biol. 1976, 68, 512-520). Tumors have been shown to have an increased rate of putrescine uptake. (Volkow, N. et al., Labeled putrescine as a probe in brain tumors. Science, 1983, 221, 673-675; and Moulinoux, J-P. et al., Biological significance of circulating polyamines in oncology. Cell. Mol. Biol. 1991, 37, 773-783).
Genes for the polyamine transport protein or complex have been cloned from Escherichia coli and yeast. (Kashiwagi, K. et al., J. Biol. Chem. 1990, 265, 20893-20897; and Tomitori, H. et al., Identification of a gene for a polyamine transport protein in yeast. J. Biol. Chem. 1999, 274, 3265-3267). The genes for the mammalian transporter await identification although Belting has hypothesized that glypican-1 may be involved. (Belting et al., Glypican-1 is a vehicle for polyamine uptake in mammalian cells: a pivital role for nitrosothiol-derived nitric oxide. J. Biol. Chem. 2003, 278(47), 47181-9). A subunit of the transporter from E. coli has been crystallized and its X-ray structure has been determined. (Sugiyama, S. et al. Crystal structure of PotD, the primary receptor of the polyamine transport system in Escherichia Coli. J. Biol. Chem. 1996, 271, 9519-9525). Since this structure was determined on a prokaryotic species, its use in the design of mammalian transport inhibitors was deemed to be of limited value.
The ability of polyamine analogs to inhibit the uptake of 3H-spermidine into cells has been studied. Bergeron and coworkers studied the effect of addition of different alkyl group substitutions on the terminal nitrogen atoms of spermidine or spermine analogs. (Bergeron, R. J. et al. Antiproliferative properties of polyamine analogs: a structure-activity study. J. Med. Chem. 1994, 37, 3464-3476).
Bergeron et al showed that larger alkyl groups diminished the ability to prevent uptake of radiolabeled spermidine. It was later concluded that increases in the number of methylenes between the nitrogen atoms decreased the ability to compete for 3H spermidine uptake. (Bergeron, R. J. et al., A comparison of structure-activity relationships between spermidine and spermine antineoplastics. J. Med. Chem. 1997, 40, 1475-1494). Bergeron et al also concluded that the polyamine transport apparatus requires only three cationic centers for polyamine recognition and transport. (Porter, C. W. et al. J. Cancer Res. 1984, 44, 126-128). Examples of the polyamine analogs' ability to inhibit 3H spermidine uptake into L1210 cells by CoMFA and QSAR methods have also been analyzed. (Li, Y. et al. Comparative molecular field analysis-based predictive model of structure-function relationships of polyamine transport inhibitors in L1210 cells. Cancer Res. 1997, 57, 234-239; and Xia, C. Q. et al. QSAR analysis of polyamine transport inhibitors in L1210 cells. J. Drug Target. 1998, 6, 65-77). A radiochemical assay is used for biochemical analysis of transport and has been used to study polyamine transport in yeast and a variety of mammalian cells. (Kakinuma, Y. et al., Biochem. Biophys. Res. Comm. 1996, 216, 985-992; and Seiler, N. et al., Int. J. Biochem. Cell Biol. 1996, 28, 843-861).
A number of series of polyamine transport inhibitors has been reported in Covassin et al., Xylylated dimers of putrescine and polyamines: influence of the polyamine backbone on spermidine transport inhibition. Bioorg Med Chem Lett. 2003, 13(19), 3267-71; Covassin et al., Synthesis of spermidine and norspermidine dimers as high affinity polyamine transport inhibitors. Bioorg Med Chem Lett. 1999, 9(12), 1709-1714; and Huber et al., 2,2′Dithiobis(N-ethyl-spermine-5-caboxamide) is a high affinity, membrane-impermeant antagonist of the mammalian polyamine transport system. J Biol. Chem. 1996, 271(44), 27556-63.
A series of amino acid spermine conjugates has been described. (Burns et al., Amino acid/spermine conjugates: polyamine amides as potent spermidine uptake inhibitors. J. Med. Chem. 2001, 44, 3632-44; Weeks et al., Exp. Cell Res. 2000, 261, 293-302; and Devens et al., Prostate Cancer and Prostatic Diseases 2000, 3, 275-279). A group of spermine dimers have been shown to be excellent polyamine uptake inhibitors. (Graminski et al., Synthesis of bis-spermine dimers that are potent polyamine transport inhibitors. Bioorg Med. Chem. Lett. 2002, 12, 35-40). Several potent polyamine transport inhibitors are disclosed in Vermeulen, N. et al., Polyamine analogues as therapeutic and diagnostic agents, U.S. Pat. No. 6,172,261 to Burns, M. R. et al., Hydrophobic polyamine analogs and methods of their use and PCT patent application WO/02053519.
Increasing concentrations of intracellular polyamine levels induce the production of antizyme which negatively regulates ODC by binding to it and targeting it for destruction. Antizyme has also been shown to inhibit polyamine uptake (Mitchell, J. L. et. al., Biochem. J. 299:19-22 (1994); Suzuki, T. et. al., Proc. Natl. Acad. Sci. USA 91: 8930-8934 (1994); Sakata, K et. al., Biochem. Biophys. Res. Commun 238:415-419 (1997)) and recent evidence suggests that antizyme may increase polyamine excretion (Sakata, K. et. al., Biochem J. 347:297-303 (2000)). Therefore, antizyme can very effectively limit the accumulation of cellular polyamines.
Antizyme has been found in vertebrates, fungi, nematodes, insects and eukaryotes (Ivanov, I. et. al., Nucleic Acids Res. 28:3185-3196 (2000)). Three antizyme isoforms, AZ1, AZ2 and AZ3, have now been identified among vertebrates. Both AZ1 and AZ2 have wide tissue distribution but AZ2 mRNA is less abundantly expressed. AZ3 is expressed only in the testis germ cells (Ivanov, I. et. al., Proc. Natl. Acad. Sci. USA 97: 4808-4813 (2000); Tosaka, Y. et. al., Genes to Cells 5:265-276 (2000)) where expression begins early in spermiogenesis and finishes in the late spermatid phase. Antizyme production is controlled by a unique regulatory mechanism known as translational frameshifting (Matsufuji; S. et. al., Cell 80: 51-60 (1995)). The antizyme gene consists of two overlapping open reading frames (ORFs). The bulk of the coding sequence is encompassed in the second (ORF2) but it does not contain an initiation codon. ORF1 is short but contains two AUG initiation codons. Either one of the initiation codons can be used to initiate translation but normally little full length mRNA is made unless a +1 frameshift occurs just before the ORF1 UGA stop codon enabling translation to continue. Only minute quantities of antizyme are generally present in mammalian tissues.
Polyamines and agmatine have been found to greatly enhance the efficiency of frameshifting (Hayashi, S. et. al., Trends Biochem. Sci. 21:27-30 (1996); Satriano, J. et. al., J. Biol. Chem. 273:15313-15316 (1998)). Vertebrates possess three elements that control frameshifling, the UGA stop codon in ORF1, a stem-loop structure 3′ to the ORF1 UGA that can base pair with a portion of the loop and conserved sequence motifs within the 3′ region of ORF1 (Matsufuji, S. et. al., Cell 80: 51-60 (1995)). It is unclear how or if polyamines interact directly with these structural elements to induce frameshifting. There may exist unknown mediators that may involve the ribosome.
In one of the first systematic assessments of antizyme induction by polyamine analogs, oligoamines such as octamines, decamines and dodecamines were found to induce antizyme to varying degrees (Mitchell, et al., Antizyme induction by polyamine analogues as a factor of cell growth inhibition, Biochem. J., 2002, 366, 663-671). These levels correlated with the cellular levels of antizyme as measured by Western blotting. A number of compounds such as bisethylnorspermine, bisethylhomospermine and 1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4) were found to induce antizyme as well as spermine. However, certain conformational restrictions within the polyamine analogs such as three, four and five-membered rings or triple bonds between the central nitrogens negatively affected antizyme induction. Many of the oligoamines greatly exceeded spermine in their ability to induce antizyme (super-induction) when tested at the same concentration (10 μM). The amount of antizyme frameshifting was found to correlate with the degree of growth inhibition. The oligoamines induced immediate cessation of cell growth, which was speculated to result from the super-induction of frameshifting. However, Mitchell et al also noted that these compounds might have other mechanisms of action leading to their observed cytotoxicity.
A number of putrescine analogs have been found to be potent reversible inhibitors of ODC. For example, 1,4-diamino-trans-2-butene inhibits ODC with a Ki of 2 μM and 1,4-phenylenediamine somewhat less potently inhibits ODC with a Ki of 46 μM (Relyea, N. et. al., Biochem. Biophys. Res. Comm. 67:392-402 (1975); Solano, F. et. al., Int. J. Biochem. 20:463-470 (1988).
A number of studies have looked at both transient and inducible overexpression of antizyme in cell lines and animal models. Anti-tumor activity was shown in a study by Iwata and colleagues (Iwata, S. et. al., Oncogene 18:165-172 (1999)) using ectopically expressed inducible antizyme. In this study, nude mice were inoculated with H-ras transformed NIH3T3 cells expressing an inducible antizyme vector. Induction of antizyme blocked tumor formation in these mice and induced cell death in vitro. Intracellular polyamine levels were also measured. Both putrescine and spermidine were completely depleted within 12 hours of induction. Spermine was also significantly reduced but over a slower time frame. Some of these observations were verified in another report that used a glucocorticoid (dexamethasone)-inducible promoter to force expression of antizyme in HZ7 cells (Murakami, Y. et. al., Biochem. J. 304:183-187 (1994)). Dexamethasone inhibited growth of this cell line, depleted putrescine levels, severely decreased spermidine levels but did not affect spermine levels. Addition of exogenous putrescine restored the intracellular putrescine levels and partially restored spermidine levels. In another study, Tsuji and colleagues (Tsuji, T. et. al., Oncogene 20:24-33 (2001)) developed a hamster malignant oral keratinocyte (HCPC-1) cell line that stably expressed antizyme. Ectopic expression of antizyme suppressed tumor mass in nude mice by about 50%. In vitro, ectopic expression significantly increased the doubling time of antizyme transfectants and the antizyme transfectants demonstrated significantly less growth in soft agar. There was also a substantial increase in Gi phase cells with a corresponding decrease in S phase cells. These cells also showed morphological alterations suggesting terminal differentiation. This was accompanied by an increase in demethylation of DNA CCGG sites of 5-methyl cytosines. It was proposed that antizyme mediates a novel mechanism in tumor suppression by reactivating key cellular genes silenced by DNA hypermethylation during cancer development. In yet another example, transgenic mice that overexpress ODC in keratinocytes have been shown to develop a high rate of spontaneous and induced skin cancer (Megosh, L. et. al., Cancer Res. 55:4205-4209 (1995)). A reduction in the frequency of induced skin-tumors was observed in the skin of these transgenic mice expressing antizyme (Feith, D. et. al. Cancer Res. 61:6073-6081 (2001)). All of these studies suggest that antizyme can reduce or inhibit tumor growth by both depleting polyamines and interfering with cell cycle progression. Examples of antizyme inducing agents can be found in Vermeulen, et al., Antizyme modulators and their use. August 2000, PCT patent WO/0046187; and Burns, M. R. Polyamine analogs that activate antizyme frameshifting, March 2004, US Pat Appl. Pub. 2004/0058954
A mutant L1210 leukemia cell line was shown to have greatly reduced polyamine transport activity following selection for resistance to methylglycoxal bis(guanylhydrazone) (MGBG), a cytotoxic AdoMetDC inhibitor that is taken up by the same transport system as the polyamines. Mice inoculated with these cells had a much greater response to DFMO treatment (87% increase in median survival time; 13 of 40 mice cured) than mice inoculated with the parental cell line (22% increase in median survival time). (Persson et al., Curative effect of d,1-2-difluoromethylornithine on mice bearing mutant L1210 leukemia cells deficient in polyamine uptake, Cancer Res. 1988, 48, 4807-4811).
A second experimental approach is based on the fact that the microbial flora in the gastrointestinal tract produces a significant source of extracellular polyamines. (Sarhan et al., The gastrointestinal tract as polyamine source for tumor growth, Anticancer Res. 1989, 9, 215-224). When this source of polyamines is removed by antibiotic treatment, DFMO's previously moderate growth inhibitory effects on Lewis lung carcinoma or L1210 cells are markedly potentiated. (Hessels et al., Limitation of dietary polyamines and arginine and the gastrointestinal synthesis of putrescine potentiates the cytostatic effect of {acute over (α)}-difluoromethylornithine in L1210 bearing mice, Int. Symp. Polyamines in Biochemical and Clinical Research, Sorrento (Italy), 1988, Abstr. P105). Finally, an additional source of polyamines is from the diet. (Bardocz et al., Polyamines in food; implications for growth and health, J. Biochem Nutr. 1993, 4, 66-71). By feeding a polyamine-free diet to DFMO-treated nude mice, MCF-7 human breast cancer xenografts contained greatly reduced levels of putrescine in comparison to DFMO treatment alone. (Levêque et al., J-Ph. The gastrointestinal polyamine source depletion enhances DFMO induced polyamine depletion in MCF-7 human breast cancer cells in vivo, Anticancer Res. 1998, 18, 2663-2668). In additional animal models, complete polyamine deprivation also enhanced DFMO's growth inhibitory effectiveness. (Moulinoux et al., Inhibition of growth of the U-251 human glioblastoma in nude mice by polyamine deprivation, Anticancer Res. 1991, 11, 175-180; Quemener et al., Polyamine deprivation enhances antitumoral efficacy of chemotherapy, Anticancer Res. 1992, 12, 1447-1454; and Chamaillard et al., Polyamine deprivation prevents the development of tumour-induced immune suppression, Br. J. Cancer 1997, 76, 365-370).
Given the importance of extracellular sources of polyamines to the growth of cells, pharmacological agents that block polyamine transport are desired. A series of simple amino acid/spermine conjugates compounds were designed, synthesized and biologically evaluated and shown to act as potent polyamine transport inhibitors in the MDA-MB-231 human breast cancer cell line. (Burns et al., Amino acid/spermine conjugates: polyamine amides as potent spermidine uptake inhibitors, J. Med. Chem. 2001, 44(22), 3632-3644). These compounds were evaluated based on their: 1) ability to inhibit the uptake of radiolabeled spermidine into MDA-MB-231 breast cancer cells; 2) their ability to increase the growth inhibitory effects of DFMO on MDA-MB-231 cells in culture even in the presence of 1 μM extracellular spermidine; 3) their inability to rescue cells from the growth inhibitory effects of DFMO in the absence of extracellular polyamines, and 4) their ability to deplete the intracellular levels of polyamines after combination treatment with DFMO. These compounds have limited cytotoxic properties when used alone, thus increasing the potential of providing tumor selectivity.
Park et.al. explored the mechanism of cell growth inhibition exerted by a series of monoguandino diamines. (Park et al, Antiproliferative effects of inhibitors of deoxyhypusine synthase, J. Bio. Chem. 1994, 269 (45), 27827-27832). Their data suggests that their effects are through the inhibition of deoxyhypusine synthase. No effects on the polyamine levels of cells were noted. N1-guanyl-1,7-diaminoheptane was shown to be the most active and its antiproliferative effects appeared to be reversible.
Additional agents, especially agmatine, are disclosed in WO 2004/078157 A2 to Oblong et al, September, 2004 Regulation of mammalian hair growth.
WO 99/03823 and its corresponding U.S. patent application Ser. No. 09/341,400, filed Jul. 6, 1999, (both of which are herby incorporated in their entireties as if fully set forth) as well as the recent publications of Burns, M. R.; Carlson, C. L.; Vanderwerf, S. M.; Ziemer, J. R.; Weeks, R. S.; Cai, F.; Webb, H. K.; Graminski, G. F. Amino acid/spermine conjugates: polyamine amides as potent spermidine uptake inhibitors, J. Med. Chem. 2001, 44, 3632-44 and Graminski, G. F.; Carlson, C. L.; Ziemer, J. R.; Cai, F., Vermeulen, N. M.; Vanerwerf, S. M.; Burns, M. R. synthesis of bis-spermine dimers that are potent polyamine transport inhibitors, Bioorg. Med. Chem. Lett. 2002, 12, 35-40 describe some extremely potent polyamine transport inhibitors.
Citation of any reference herein is not intended as an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these documents.